CN101715731A - Method for culturing Euramerican poplar tissues to regenerate Euramerican poplar - Google Patents
Method for culturing Euramerican poplar tissues to regenerate Euramerican poplar Download PDFInfo
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- CN101715731A CN101715731A CN200910311186A CN200910311186A CN101715731A CN 101715731 A CN101715731 A CN 101715731A CN 200910311186 A CN200910311186 A CN 200910311186A CN 200910311186 A CN200910311186 A CN 200910311186A CN 101715731 A CN101715731 A CN 101715731A
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Abstract
The invention relates to a method for culturing Euramerican poplar tissues to regenerate the Euramerican poplar, comprising the steps of obtaining sterile explants, inducing and differentiating calluses, stooling clustered sprouts, raising clustered seedlings and culturing the clustered seedlings to make the clustered seedlings root. The method for culturing Euramerican poplar tissues to regenerate the Euramerican poplar is easy to operate and control, can be used for repeatedly producing the Euramerican poplar annually and can effectively meet the need of studying and culturing the Euramerican poplar, ensure the highest adventitious sprout inducing rate of 100 percent and the clustered seedling rooting rate of more than 96.7 percent and improve the Euramerican poplar regenerating rate by culturing the Euramerican poplar tissues to a great extent.
Description
Technical field
The present invention relates to a kind of method of culturing Euramerican poplar tissues to regenerate Euramerican poplar, belong to field of plant tissue culture technique.
Background technology
The willow gene engineering of China has obtained many achievements, but how the transgene receptor willow is not main breed, existing its fast natural disposition of transgenic poplar and adaptability all are subjected to the limitation of genetic prerequisite, many gene engineering achievements in research fail extensive use in production of forestry (referring to the Lu Meng post build up the Army recklessly China's transgenic poplar of in the forestry science and technology exploitation, delivering in 2006 research and use present situation).More combine closely with the production of forestry practical application in order to make willow gene engineering improvement research, the genetic transformation of some important main breeds is broken through and is had great realistic meaning from produce, and tame in the world at present Populus kind has that all to come from black poplar group more than 90% be American-European poplar.American-European poplar 2001 adaptability are strong, all have superiority at aspects such as cold-resistant, drought-enduring, disease-resistant worms, suit extensively to plant at western part of China and North China; American-European poplar 2001 is a kind of desirable transgenic acceptors, so the system of tissue culture regeneration efficiently of explant successfully sets up, and its biological study, genetic improvement and Study on Genetic Transformation work are laid a good foundation.
Summary of the invention
The object of the present invention is to provide a kind of method of culturing Euramerican poplar tissues to regenerate Euramerican poplar.
Technical program of the present invention lies in adopting a kind of method of culturing Euramerican poplar tissues to regenerate Euramerican poplar, this method may further comprise the steps:
1. the acquisition of aseptic explant: with American-European Yang Jiankang spray is material, and the petiole cleaning and sterilizing is obtained aseptic petiole explant;
2. callus induce differentiation: aseptic petiole is inoculated on the inducing culture, begins to differentiate a large amount of indefinite buds after two weeks, 14-20 days successive transfer culture are once;
3. put forth and strong sprout: the bud of will growing thickly is transferred on the medium of putting forth together with explant, put forth be cultured to the bud of growing thickly and be higher than 2cm after, the seedling of will growing thickly is transferred on the strong seedling culture base, makes the seedling of growing thickly be grown to the no offspring of stem stalwartness;
4. culture of rootage: will not have the offspring individual plant and be inoculated in the root media, and cultivate that to obtain main root sturdy, the seedling that lateral root is abundant.
Described inducing culture is MS+TDZ0.025-0.1mg/L+6-BA0.025-0.1mg/L+NAA0.02-0.1mg/L+ sucrose 20-40g/L.
The described medium of putting forth is MS+KT0.5-1mg/L+GA
31-4mg/L+ fructose 20-40g/L.
Described strong seedling culture base does not contain the MS+ sucrose 20-40g/L of plant hormone.
Described root media: 1/2MS+IBA0.02-0.1mg/L+IAA0.02-0.1mg/L+ active carbon 1-5mg/L+ sucrose 20-40g/L.
All medium all contain the agar of 4-6g/L, and the pH value is 5.6-6.2.
The cultivation temperature of described tissue culture should be controlled at 24-26 ℃, and intensity of illumination is 1500-2000lx, and light application time is 12-16 hour.
The method of described petiole cleaning and sterilizing comprises petiole after the running water that flows washes down, places superclean bench 70%-75% Ethanol Treatment 30-50s, and aseptic water washing is removed residue; With 0.5-1g/L mercuric chloride solution sterilization 3-6 minute, remove residue with aseptic water washing at last again, obtain aseptic petiole explant.
American-European poplar used in the present invention is American-European poplar 2001.
The advantage of the inventive method is: incubation is convenient to operation and control, but annual requirement to the willow tissue cultivating seedling can be effectively satisfied in the anniversary duplication of production; And the inductivity of indefinite bud reaches as high as 100% in the inventive method, and rooting rate reaches more than 96.7%, has improved the efficient that obtains regeneration plant by tissue culture.
Description of drawings
Each growth period situation schematic diagram of regeneration plant that Fig. 1 obtains for the inventive method, a is the bud of growing thickly, and b is the bud of growing thickly of putting forth, and c is the bud of growing thickly after strong sprout, and d is the seedling after taking root.
Embodiment
The method of present embodiment culturing Euramerican poplar tissues to regenerate Euramerican poplar may further comprise the steps
1. the acquisition of aseptic explant: with newborn healthy spray on the American-European poplar 2001 annotinous branch cuttage seeding is material, in the running water that flows down after the flushing, places superclean bench with 70% Ethanol Treatment 30s petiole, and aseptic water washing is removed residue; With 1g/L mercuric chloride solution sterilization 5min, remove residue with aseptic water washing at last again, obtain aseptic petiole explant;
2. callus induce differentiation: aseptic petiole is inoculated in inducing culture (MS+TDZ0.05mg/L+6-BA0.05mg/L+NAA0.05mg/L+ sucrose 20g/L+ agar 5g/L, the pH value is 5.8) on, notch reddens and expands behind the 5d, begin to form green callus, begin to occur a large amount of indefinite buds behind the 14d on the callus, bud acaulescence (the Fig. 1-a) of growing thickly, subculture is once on inducing culture every 15d, inoculate 120 aseptic petiole sections altogether, all petioles all can be induced and be differentiated the bud of growing thickly, and adventitious bud induction frequency reaches 100%;
3. put forth and strong sprout: the bud of growing thickly is transferred to the medium (MS+KT0.5mg/L+GA that puts forth together with former explant
32mg/L+ fructose 20g/L+ agar 5g/L, the pH value is 5.8) on, the cultivation of putting forth, the thin and delicate (Fig. 1-b) of the bud of growing thickly; After bud to be grown thickly was higher than 2cm, the seedling of will growing thickly was transferred on the strong seedling culture base (MS+ sucrose 200g/L+ agar 5g/L, the pH value is 5.8), made the seedling of growing thickly be grown to no offspring (Fig. 1-c) of stem stalwartness;
4. culture of rootage: select 4～6cm height, the no offspring that the leaf look normal, stem is sturdy to be inoculated into root media (1/2MS+IBA0.1mg/L+IAA 0.02mg/L+ active carbon 2mg/L+ sucrose 200g/L+ agar 5g/L, the pH value is 5.8) on, red sturdy point appears in the seedling base portion behind the inoculation 7d, root reaches about 4cm behind the 20d, main root is sturdy, and lateral root quantity is enriched (Fig. 1-d), inoculate 50 no offspring altogether, wherein take root for 49, rooting rate reaches 98%.
Wherein the cultivation temperature of tissue culture should be controlled at 25 ℃, and intensity of illumination is 2000lx, and light application time is 15 hours.
Embodiment 2
The method of present embodiment culturing Euramerican poplar tissues to regenerate Euramerican poplar may further comprise the steps
1. the acquisition of aseptic explant: with American-European Yang Jiankang spray is material, in the running water that flows down after the flushing, places superclean bench with 70% Ethanol Treatment 30s petiole, and aseptic water washing is removed residue; With 0.5g/L mercuric chloride solution sterilization 6min, remove residue with aseptic water washing at last again, obtain aseptic petiole explant;
2. callus induce differentiation: aseptic petiole is inoculated in inducing culture (MS+TDZ0.025mg/L+6-BA0.1mg/L+NAA 0.02mg/L+ sucrose 20g/L+ agar 6g/L, the pH value is 5.6) on, begin to differentiate a large amount of indefinite buds after two weeks, 14 days successive transfer culture once, inoculate 120 aseptic petiole sections altogether, wherein 109 petioles can be induced and be differentiated the bud of growing thickly, and adventitious bud induction frequency reaches 91%;
3. put forth and strong sprout: the bud of will growing thickly is transferred to the medium (MS+KT1mg/L+GA that puts forth together with explant
31mg/L+ fructose 20g/L+ agar 6g/L, the pH value is 5.6) on, put forth be cultured to the bud of growing thickly and be higher than 2cm after, the seedling of will growing thickly is transferred on the strong seedling culture base (MS+ sucrose 40g/L+ agar 4g/L, the pH value is 5.6), makes the seedling of growing thickly be grown to the no offspring of stem stalwartness;
4. culture of rootage: select 4～6cm height, the no offspring that the leaf look normal, stem is sturdy to be inoculated into root media (1/2MS+IBA 0.02mg/L+IAA0.1mg/L+ active carbon 1mg/L+ sucrose 40g/L+ agar 4g/L, the pH value is 5.6) in, red sturdy point appears in the seedling base portion behind the inoculation 7d, root reaches about 4cm behind the 20d, main root is sturdy, and the seedling that lateral root is abundant inoculates 60 no offspring altogether, wherein take root for 58, rooting rate reaches 96.7%.
Wherein the cultivation temperature of tissue culture should be controlled at 24 ℃, and intensity of illumination is 1500lx, and light application time is 16 hours.
Embodiment 3
The method of present embodiment culturing Euramerican poplar tissues to regenerate Euramerican poplar may further comprise the steps
1. the acquisition of aseptic explant: with American-European Yang Jiankang spray is material, in the running water that flows down after the flushing, places superclean bench with 75% Ethanol Treatment 50s petiole, and aseptic water washing is removed residue; With 1g/L mercuric chloride solution sterilization 3min, remove residue with aseptic water washing at last again, obtain aseptic petiole explant;
2. callus induce differentiation: aseptic petiole is inoculated in inducing culture (MS+TDZ0.1mg/L+6-BA0.025mg/L+NAA 0.1mg/L+ sucrose 40g/L+ agar 4g/L, the pH value is 6.2) on, begin to differentiate a large amount of indefinite buds after two weeks, 20 days successive transfer culture once, inoculate 120 aseptic petiole sections altogether, wherein 112 petioles can be induced and be differentiated the bud of growing thickly, and adventitious bud induction frequency reaches 93%;
3. put forth and strong sprout: the bud of will growing thickly is transferred to the medium (MS+KT0.5mg/L+GA that puts forth together with explant
34mg/L+ fructose 40g/L+ agar 4g/L, the pH value is 6.2) on, put forth be cultured to the bud of growing thickly and be higher than 2cm after, the seedling of will growing thickly is transferred on the strong seedling culture base (MS+ sucrose 20g/L+ agar 6g/L, the pH value is 6.2), makes the seedling of growing thickly be grown to the no offspring of stem stalwartness;
4. culture of rootage: select 4～6cm height, the no offspring that the leaf look normal, stem is sturdy to be inoculated into root media (1/2MS+IBA0.1mg/L+IAA 0.02mg/L+ active carbon 5mg/L+ sucrose 20g/L+ agar 6g/L, the pH value is 6.2) in, red sturdy point appears in the seedling base portion behind the inoculation 7d, root reaches about 4cm behind the 20d, main root is sturdy, the seedling that lateral root is abundant inoculates 40 no offspring altogether, wherein takes root for 39; Rooting rate reaches 97.5%.
Wherein the cultivation temperature of tissue culture should be controlled at 26 ℃, and intensity of illumination is 2000lx, and light application time is 12 hours.
Claims (8)
1. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar is characterized in that: said method comprising the steps of
1. the acquisition of aseptic explant: with American-European Yang Jiankang spray is material, and the petiole cleaning and sterilizing is obtained aseptic petiole explant;
2. callus induce differentiation: aseptic petiole is inoculated on the inducing culture, begins to differentiate a large amount of indefinite buds after two weeks, 14-20 days successive transfer culture are once;
3. put forth and strong sprout: the bud of will growing thickly is transferred on the medium of putting forth together with explant, put forth be cultured to the bud of growing thickly and be higher than 2cm after, the seedling of will growing thickly is transferred on the strong seedling culture base, makes the seedling of growing thickly be grown to the no offspring of stem stalwartness;
4. culture of rootage: will not have the offspring individual plant and be inoculated in the root media, and cultivate that to obtain main root sturdy, the seedling that lateral root is abundant.
2. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1, it is characterized in that: described inducing culture is MS+TDZ 0.025-0.1mg/L+6-BA 0.025-0.1mg/L+NAA0.02-0.1mg/L+ sucrose 20-40g/L.
3. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1, it is characterized in that: the described medium of putting forth is MS+KT 0.5-1mg/L+GA3 1-4mg/L+ fructose 20-40g/L.
4. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1, it is characterized in that: described strong seedling culture base does not contain the MS+ sucrose 20-40g/L of plant hormone.
5. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1 is characterized in that: described root media: 1/2MS+IBA 0.02-0.1mg/L+IAA 0.02-0.1mg/L+ active carbon 1-5mg/L+ sucrose 20-40g/L.
6. according to the method for any described culturing Euramerican poplar tissues to regenerate Euramerican poplar among the claim 2-5, it is characterized in that: described medium all contains the agar of 4-6g/L, and the pH value is 5.6-6.2.
7. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1, it is characterized in that: the cultivation temperature of described tissue culture should be controlled at 24-26 ℃, and intensity of illumination is 1500-2000lx, and light application time is 12-16 hour.
8. the method for culturing Euramerican poplar tissues to regenerate Euramerican poplar according to claim 1, it is characterized in that: the method for described petiole cleaning and sterilizing comprises that petiole is after the running water that flows washes down, place superclean bench 70%-75% Ethanol Treatment 30-50s, aseptic water washing is removed residue; With 0.5-1g/L mercuric chloride solution sterilization 3-6min, remove residue with aseptic water washing at last again, obtain aseptic petiole explant.
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| CN102550382A (en) * | 2012-03-20 | 2012-07-11 | 东北林业大学 | Rooting transplanting method for tissue culture poplar seedlings |
| CN102668979A (en) * | 2012-01-10 | 2012-09-19 | 河南科技大学 | Rooting culture method for poplar tissue culture seedlings |
| CN102919128A (en) * | 2012-11-14 | 2013-02-13 | 东北林业大学 | Out-of-bottle bag rooting method for tissue culture seedlings of Europe and America hybrid aspens |
| CN103468717A (en) * | 2013-09-25 | 2013-12-25 | 大连民族学院 | Populus deltoids*populus nigra basic-domain leucine-zipper (PdbZIP) gene and application thereof |
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| CN108575740A (en) * | 2018-02-26 | 2018-09-28 | 北京林业大学 | A kind of method that the generation of willow body embryo is built up with plant |
| CN110583489A (en) * | 2019-10-24 | 2019-12-20 | 南京林业大学 | Tissue culture rapid propagation method and application of populus euphratica |
| CN112335550A (en) * | 2020-12-23 | 2021-02-09 | 江苏省中国科学院植物研究所 | Method for establishing regeneration system of populus colourianus by taking petioles as explants |
| CN113748989A (en) * | 2021-10-26 | 2021-12-07 | 山东省林业科学研究院 | Method for inducing somatic embryo of in vitro leaf of 107 populus deltoids |
| CN115088621A (en) * | 2022-07-18 | 2022-09-23 | 南京林业大学 | Method for promoting massive and rapid differentiation of poplar explants into buds by vacuum pumping treatment |
| CN116548313A (en) * | 2023-06-27 | 2023-08-08 | 苏州北美国际高级中学 | Method for in-vitro root induction regeneration of Euramerican poplar hybrid tissue culture seedlings |
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2009
- 2009-12-10 CN CN2009103111861A patent/CN101715731B/en not_active Expired - Fee Related
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| CN102668979A (en) * | 2012-01-10 | 2012-09-19 | 河南科技大学 | Rooting culture method for poplar tissue culture seedlings |
| CN102668979B (en) * | 2012-01-10 | 2014-05-21 | 河南科技大学 | Rooting culture method for poplar tissue culture seedlings |
| CN102550382B (en) * | 2012-03-20 | 2013-06-19 | 东北林业大学 | Rooting transplanting method for tissue culture poplar seedlings |
| CN102550382A (en) * | 2012-03-20 | 2012-07-11 | 东北林业大学 | Rooting transplanting method for tissue culture poplar seedlings |
| CN102919128A (en) * | 2012-11-14 | 2013-02-13 | 东北林业大学 | Out-of-bottle bag rooting method for tissue culture seedlings of Europe and America hybrid aspens |
| CN103468717B (en) * | 2013-09-25 | 2014-12-10 | 大连民族学院 | Populus deltoids*populus nigra basic-domain leucine-zipper (PdbZIP) gene and application thereof |
| CN103468717A (en) * | 2013-09-25 | 2013-12-25 | 大连民族学院 | Populus deltoids*populus nigra basic-domain leucine-zipper (PdbZIP) gene and application thereof |
| CN103636498B (en) * | 2013-11-29 | 2015-12-02 | 黑龙江省林业科学研究所 | Aspen breeding silver × mountain 1333 tissue culture medium (TCM) |
| CN103636498A (en) * | 2013-11-29 | 2014-03-19 | 黑龙江省林业科学研究所 | Populus alba * pobulus davidiana dode 1333 tissue culture medium of pobulus davidiana dode improved variety |
| CN108575740A (en) * | 2018-02-26 | 2018-09-28 | 北京林业大学 | A kind of method that the generation of willow body embryo is built up with plant |
| CN108575740B (en) * | 2018-02-26 | 2020-12-01 | 北京林业大学 | Method for generating poplar somatic embryo and building plant |
| CN110583489A (en) * | 2019-10-24 | 2019-12-20 | 南京林业大学 | Tissue culture rapid propagation method and application of populus euphratica |
| CN112335550A (en) * | 2020-12-23 | 2021-02-09 | 江苏省中国科学院植物研究所 | Method for establishing regeneration system of populus colourianus by taking petioles as explants |
| CN113748989A (en) * | 2021-10-26 | 2021-12-07 | 山东省林业科学研究院 | Method for inducing somatic embryo of in vitro leaf of 107 populus deltoids |
| CN115088621A (en) * | 2022-07-18 | 2022-09-23 | 南京林业大学 | Method for promoting massive and rapid differentiation of poplar explants into buds by vacuum pumping treatment |
| CN115088621B (en) * | 2022-07-18 | 2023-09-26 | 南京林业大学 | Method for promoting mass and rapid differentiation of poplar explants into buds by utilizing vacuumizing treatment |
| CN116548313A (en) * | 2023-06-27 | 2023-08-08 | 苏州北美国际高级中学 | Method for in-vitro root induction regeneration of Euramerican poplar hybrid tissue culture seedlings |
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